1. Field of the Invention
This invention relates to and has among its objects novel non-toxic immunizing compositions against gram-negative bacteria and novel methods for their preparation. It is a particular object of the invention to prepare an immunizing composition effective against Pseudomonas aeruginosa infections. Further objects of the invention will be evident from the following description wherein parts and percentages are by weight unless otherwise specified.
2. Description of the Prior Art
Gram-negative bacteria have two cell envelope membranes separated by a single thin layer of peptidoglycan. The inner or cytoplasmic membrane contains all known active transport systems and many of the cell envelop enzymes. The outer membrane is distinguished by a unique component lipopolysaccharide (lipid A plus polysaccharide) and a unique set of proteins. The O antigen-specific polysaccharide endows the particular bacterium with its main serological specificity. There is also a core polysaccharide, common to gram-negative bacteria, which is linked to a lipid component (lipid A). These complexes of lipid A, polysaccharide, and protein are antigenic and also exert toxic reactions in humans, thus being considered as endotoxins.
Vaccines containing lipopolysaccharides (LPS) from gram-negative bacteria have been used for immunization of humans against infection. The vaccines may comprise killed cells, cell lysate, or purified LPS; however, many are toxic.
Although infection with Pseudomonas aeruginosa (P. aeruginosa) is not common among the general population, P. aeruginosa infection is encountered very frequently in certain susceptible groups of patients. Burn victims and immunosuppressed cancer patients have been identified as having an unusually high risk of acquiring severe, and sometimes fatal, P. aeruginosa infection. P. aeruginosa infections are usually acquired during a hospital stay, not at home.
Antibiotics have been used to treat patients with P. aeruginosa infections. However, antibiotic treatment is very expensive, effectiveness is often uncertain, and organisms continue to develop resistance to antibiotics.
Vaccines have been prepared for a number of pathogenic bacteria, including P. aeruginosa. For example, U.S. Pat. No. 4,157,389 discloses a three component mixed vaccine against infections caused by P. aeruginosa which comprises as the antigens an infection-protective common antigen, Original Endotoxin Protein obtained from P. aeruginosa, an elastase toxoid obtained from P. aeruginosa and a protease toxoid obtained from P. aeruginosa.
Toxoids derived from protease and elastase of P. aeruginosa which are effective to prevent infections caused by P. aeruginosa are described in U.S. Pat. No. 4,160,023.
U.S. Pat. No. 3,987,164 discloses vaccine preparations comprising the cell wall protein component of P. aeruginosa as an active ingredient in a prophylactic pharmaceutical preparation.
Mink infection caused by P. aeruginosa can be prevented according to U.S. Pat. No. 4,096,245 by administering to mink a prophylactic preparation in the form of vaccine whose effective component mainly consists of protein and a small amount of lipid and sugar derived from P. aeruginosa.
Original endotoxin protein derived from P. aeruginosa is disclosed in U.S. Pat. No. 4,079,126. In the patented method of preparation the original endotoxin protein is processed with either proteolytic enzyme or reductant or further processed with proteolytic enzyme after it has been treated with reductant.
A bacterial endotoxin LPS of reduced toxicity covalently coupled to a protein antigen is described in U.S. Pat. No. 4,185,090. The coupling was effected by reaction with haloacylhalide. LPS acylated with an anhydride of a dibasic acid is detoxified; in combination with endotoxin polysaccharide covalently coupled to protein antigen it developed synergistic immunogenic effects.
In U.S. Pat. No. 4,285,936 a method is taught for isolating a non-toxic, high molecular weight polysaccharide antigen from the crude slime of a P. aeruginosa culture, and a method for inducing immunity in a host to said live organisms is described. Initially, bacterial cells are separated from the slime, which is dissolved in a phosphate buffer solution. After removal of dissolved contaminating nucleic acids, a lipid A portion of the contaminating LPS constituent is removed and precipitated by acetic acid hydrolysis. The remaining lipids are extracted with chloroform. Nearly all of the residual nucleic acids are then removed by digestion with nucleases, and the remaining protein extracted with phenol. The aqueous and phenol layers are separated, and the aqueous layer applied to a gel filter to isolate the polysaccharide antigen by column chromatography. The polysaccharide antigen was non-toxic and highly effective in inducing an immune response to the organism in a host.